Cellulose is relied upon as the raw material for a number of useful products including paper products and wound dressings. Cellulose may be obtained from plants and various microorganisms in culture, for example from the cellulose producing bacteria of the genus Acetobacter. Acetobacter is characteristically a Gram-negative, rod-shaped bacterium 0.6-0.8 .mu.m by 1.0-4 .mu.m. The organism is strictly aerobic; metabolism is respiratory, never fermentative. It is further distinguished by the ability to produce multiple poly .beta.(1-4)-glucan chains, chemically identical to cellulose. Multiple cellulose chains or microfibrils are synthesized at the bacterial surface at sites on the cell wall. The production of cellulose by Acetobacter has been the subject of intense study since at least the 1930's. In particular, Acetobacter xylinum has been widely studied to attempt to elucidate the mechanism of cellulose synthesis in intact cells [Schramm and Hestrin, (1954) J. Gen. Microbiol. 11:123-129].
The enzymatic pathway for cellulose synthesis in Acetobacter xylinum has been investigated and essentially four enzymatic steps have been characterized in cell-free extracts of A. xylinum which appear to comprise the complete pathway from glucose to cellulose. These are the phosphorylation of glucose by glucokinase [Benziman, et al., (1972) J. Bacteriol., 111:325-330], the isomerization of glucose-6-phosphate to glucose-1-phosphate by phosphoglucomutase [Gromet, et al., (1957) Biochem. J., 67:67914 689; Frei-Roitman, Factors affecting the activity of phosphoglucomutase and UDP-glucose pyrophosphorylase of Acetobacter xylinum, M.Sc. thesis, The Hebrew University of Jerusalem, Jerusalem, Israel (1974)]; the synthesis of uridine 5'-diphosphoglucose (UDP-glc) by UDPG-pyrophosphorylase, [Frei-Roitman, supra; Swissa, Biosynthesis of cellulose in Acetobacter xylinum, Ph.D. thesis, The Hebrew University of Jerusalem, Jerusalem, Israel (1978)], and the cellulose synthase reaction.
A detailed review of the genetics and biochemistry of cellulose synthesis in Acetobacter and other cellulose producing bacteria is provided in Ross, et al., Microbiological Reviews, 55:35-38 (1991) .
Early attempts to purify cellulose synthase from a strain of A. xylinum employing conventional chromatographic techniques have not been especially successful, but recently the enzyme has been significantly purified and its properties and structure in the purified state have been investigated (Mayer et al., Proc. Natl. Acad. Sci. USA 88:5472-5476 (1991).
Similarly, attempts to purify cellulose synthase by in vitro cellulose entrapment and chromatographic techniques have resulted in a partially purified 83 kilodalton (kD) polypeptide (Lin and Brown, in Cellulose and Wood-Chemistry and Technology (1989) ED. Scharch, Wiley, N.Y. pp.473-492).
A more complete knowledge of the biochemistry of cellulose synthesis would facilitate greater productivity and yield of cellulose from cultures of cellulose-producing microorganisms.
The formation of cellulose, i.e., the polymerization of glucose, appears to be catalyzed by the gene product of the bcsA gene product of the bcs (bacterial cellulose synthase operon). The bcsA gene product appears to be physically associated with a regulatory subunit that is the product of the bcsB gene. Mutations in the other bcs operon genes have shown that these genes are also important for cellulose synthesis, as described in Wong et al. Proc. Natl. Acad. Sci. 87:8130-8134 (1990).
In addition to the bcsA, bcsB, bcsC and bcsD gene products of the bcs operon, several other proteins may be involved in the production of cellulose in vivo. The identification of these additional proteins may be useful in regulating cellulose production by recombinant micro-organisms because, among other reason, altering the expressive level of these proteins in cellulose producing bacteria may be used to modulate the production of cellulose. Some of these proteins may physically interact with cellulose synthase. Those proteins capable of interacting with cellulose synthase have a variety of uses in addition to their biological activity. These biological activity independent uses of cellulose synthase interacting proteins are based, at least in part, on the specific interaction between the cellulose synthase interacting proteins and cellulose synthase.